Although methotrexate is a potent (nearly stoichiometric) inhibitor of dihydrofolate reductase, additional properities are needed of folate antagonists to overcome acquired resistance to initial chemotherapy, and to combat slow growing solid tumors. In designing and testing new antifolates consideration should be given to their possible interference with other pteridine utilizing systems. This research project is aimed at determining the molecular basis of specificity of a class of enzymes which use a pteridine cofactor, tetrahydrobiopterin, so that in the development of new folate antagonists toxic side effects can be minimized. A series of pteridines and pteridine-like compounds, diverging systematically from the structure of biopterin, is being used to probe the requiremnts for binding and for cofactor activity with phenylalanine hydroxylase and tryptophan and tyrosine hydroxylases, which catalyze the biosynthesis of the neurotransmitters dopa, epinephrine and serotonin. The effects of the pteridine compounds are also being tested on (1) dihydropteridine reductase, the enzyme which regenerates tetrahydrobiopterin after reaction with the aromatic amino acid hydroxylases, and (2) dihydrofolate reductase which, in addition to its role in folate metabolism, provides the only known mechanism for reducing the dietary form of biopterin to tetrahydrobiopterin. The structure-action relationships determined in this work will aid in the interpretation of drug screening, and will provide the information necessary to make selective inhibitors of dihydrofolate reductase, thereby allowing extension of the pharmacological dimensions of antifolates.